New Comparative density of CCK- and PV-GABA cells within the … · 2017. 4. 13. · Whissell et...

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ORIGINAL RESEARCH published: 23 September 2015 doi: 10.3389/fnana.2015.00124 Edited by: Yun-Qing Li, The Fourth Military Medical University, China Reviewed by: Jean-Pierre Hornung, University of Lausanne, Switzerland Frasncisco E. Olucha-Bordonau, University of Valencia, Spain *Correspondence: Jun Chul Kim, Department of Psychology, University of Toronto, 100 Saint George Street, Toronto, ON M5S 3G3, Canada [email protected] Received: 06 July 2015 Accepted: 31 August 2015 Published: 23 September 2015 Citation: Whissell PD, Cajanding JD, Fogel N and Kim JC (2015) Comparative density of CCK- and PV-GABA cells within the cortex and hippocampus. Front. Neuroanat. 9:124. doi: 10.3389/fnana.2015.00124 Comparative density of CCK- and PV-GABA cells within the cortex and hippocampus Paul D. Whissell 1 , Janine D. Cajanding 2 , Nicole Fogel 2 and Jun Chul Kim 1,2 * 1 Department of Psychology, University of Toronto, Toronto, ON, Canada, 2 Cell and Systems Biology, University of Toronto, Toronto, ON, Canada Cholecystokinin (CCK)- and parvalbumin (PV)-expressing neurons constitute the two major populations of perisomatic GABAergic neurons in the cortex and the hippocampus. As CCK- and PV-GABA neurons differ in an array of morphological, biochemical and electrophysiological features, it has been proposed that they form distinct inhibitory ensembles which differentially contribute to network oscillations and behavior. However, the relationship and balance between CCK- and PV-GABA neurons in the inhibitory networks of the brain is currently unclear as the distribution of these cells has never been compared on a large scale. Here, we systemically investigated the distribution of CCK- and PV-GABA cells across a wide number of discrete forebrain regions using an intersectional genetic approach. Our analysis revealed several novel trends in the distribution of these cells. While PV-GABA cells were more abundant overall, CCK-GABA cells outnumbered PV-GABA cells in several subregions of the hippocampus, medial prefrontal cortex and ventrolateral temporal cortex. Interestingly, CCK-GABA cells were relatively more abundant in secondary/association areas of the cortex (V2, S2, M2, and AudD/AudV) than they were in corresponding primary areas (V1, S1, M1, and Aud1). The reverse trend was observed for PV-GABA cells. Our findings suggest that the balance between CCK- and PV-GABA cells in a given cortical region is related to the type of processing that area performs; inhibitory networks in the secondary cortex tend to favor the inclusion of CCK-GABA cells more than networks in the primary cortex. The intersectional genetic labeling approach employed in the current study expands upon the ability to study molecularly defined subsets of GABAergic neurons. This technique can be applied to the investigation of neuropathologies which involve disruptions to the GABAergic system, including schizophrenia, stress, maternal immune activation and autism. Keywords: cholecystokinin, parvalbumin, interneuron, cortex, hippocampus, Dlx genes, intersectional genetics, balance Introduction Inhibitory neurotransmission shapes neuronal activity and thereby regulates information processing (Freund, 2003). In the central nervous system, inhibition is predominantly mediated by interneurons signaling through the transmitter γ-aminobutyric acid (GABA; Kepecs and Fishell, 2014). Though interneurons constitute only a fraction of all neurons (15%) (Beaulieu, 1993), Frontiers in Neuroanatomy | www.frontiersin.org 1 September 2015 | Volume 9 | Article 124 CORE Metadata, citation and similar papers at core.ac.uk Provided by Frontiers - Publisher Connector

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Page 1: New Comparative density of CCK- and PV-GABA cells within the … · 2017. 4. 13. · Whissell et al. Comparative density of CCK- and PV-GABA cells they are indispensable for multiple

ORIGINAL RESEARCHpublished: 23 September 2015

doi: 10.3389/fnana.2015.00124

Edited by:Yun-Qing Li,

The Fourth Military Medical University,China

Reviewed by:Jean-Pierre Hornung,

University of Lausanne, SwitzerlandFrasncisco E. Olucha-Bordonau,

University of Valencia, Spain

*Correspondence:Jun Chul Kim,

Department of Psychology, Universityof Toronto, 100 Saint George Street,

Toronto, ON M5S 3G3, [email protected]

Received: 06 July 2015Accepted: 31 August 2015

Published: 23 September 2015

Citation:Whissell PD, Cajanding JD, Fogel N

and Kim JC (2015) Comparativedensity of CCK- and PV-GABA cellswithin the cortex and hippocampus.

Front. Neuroanat. 9:124.doi: 10.3389/fnana.2015.00124

Comparative density of CCK- andPV-GABA cells within the cortex andhippocampusPaul D. Whissell1, Janine D. Cajanding2, Nicole Fogel2 and Jun Chul Kim1,2*

1 Department of Psychology, University of Toronto, Toronto, ON, Canada, 2 Cell and Systems Biology, University of Toronto,Toronto, ON, Canada

Cholecystokinin (CCK)- and parvalbumin (PV)-expressing neurons constitute thetwo major populations of perisomatic GABAergic neurons in the cortex and thehippocampus. As CCK- and PV-GABA neurons differ in an array of morphological,biochemical and electrophysiological features, it has been proposed that they formdistinct inhibitory ensembles which differentially contribute to network oscillations andbehavior. However, the relationship and balance between CCK- and PV-GABA neuronsin the inhibitory networks of the brain is currently unclear as the distribution of thesecells has never been compared on a large scale. Here, we systemically investigatedthe distribution of CCK- and PV-GABA cells across a wide number of discrete forebrainregions using an intersectional genetic approach. Our analysis revealed several noveltrends in the distribution of these cells. While PV-GABA cells were more abundantoverall, CCK-GABA cells outnumbered PV-GABA cells in several subregions of thehippocampus, medial prefrontal cortex and ventrolateral temporal cortex. Interestingly,CCK-GABA cells were relatively more abundant in secondary/association areas of thecortex (V2, S2, M2, and AudD/AudV) than they were in corresponding primary areas (V1,S1, M1, and Aud1). The reverse trend was observed for PV-GABA cells. Our findingssuggest that the balance between CCK- and PV-GABA cells in a given cortical region isrelated to the type of processing that area performs; inhibitory networks in the secondarycortex tend to favor the inclusion of CCK-GABA cells more than networks in the primarycortex. The intersectional genetic labeling approach employed in the current studyexpands upon the ability to study molecularly defined subsets of GABAergic neurons.This technique can be applied to the investigation of neuropathologies which involvedisruptions to the GABAergic system, including schizophrenia, stress, maternal immuneactivation and autism.

Keywords: cholecystokinin, parvalbumin, interneuron, cortex, hippocampus, Dlx genes, intersectional genetics,balance

Introduction

Inhibitory neurotransmission shapes neuronal activity and thereby regulates informationprocessing (Freund, 2003). In the central nervous system, inhibition is predominantly mediated byinterneurons signaling through the transmitter γ-aminobutyric acid (GABA; Kepecs and Fishell,2014). Though interneurons constitute only a fraction of all neurons (∼15%) (Beaulieu, 1993),

Frontiers in Neuroanatomy | www.frontiersin.org 1 September 2015 | Volume 9 | Article 124

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Provided by Frontiers - Publisher Connector

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Whissell et al. Comparative density of CCK- and PV-GABA cells

they are indispensable for multiple behavioral functions,including fear, anxiety, social interaction, olfaction, locomotionand memory (Cho et al., 2013; Donato et al., 2013; Courtinet al., 2014; Wolff et al., 2014). Imbalances in interneuronsignaling have serious consequences for behavior, and have beenimplicated in the pathogenesis of neuropsychiatric disorderssuch as schizophrenia, Huntington’s disease, epilepsy andAlzheimer’s disease (Peng et al., 2013; Perez and Lodge, 2013;Aldrin-Kirk et al., 2014; Godavarthi et al., 2014; Kim et al.,2014a; Knoferle et al., 2014; Ma and McLaurin, 2014; Tonget al., 2014; Zamberletti et al., 2014). Collectively, interneuronsrepresent a heterogeneous group of cells which differ inmorphology, molecular composition, electrophysiologicalproperties and distribution within the central nervous system(Kepecs and Fishell, 2014). Each of these interneuron subtypesmay differentially contribute to behavioral function in health anddisease (Lapray et al., 2012; Nguyen et al., 2014), and it remainsa major challenge for neuroscience to characterize their specificproperties.

Interneurons can be classified according to the targetingof their axonal projections to post-synaptic cells. Perisomaticinterneurons, which account for nearly half of all hippocampaland cortical interneurons, form inhibitory synapses near thecell soma, proximal dendrites, and axon initial segments oftheir targets (Freund and Buzsaki, 1996). Perisomatic inhibitionefficiently suppresses repetitive sodium-dependent actionpotentials (Miles et al., 1996) and entrains the networkoscillations (Buzsaki and Wang, 2012) that accompanyhigher order cognitive operations (Wang, 2010). Perisomaticinterneurons can be further subdivided into two separategroups based on the expression of the molecular markerscholecystokinin (CCK-GABA neurons) and parvalbumin(PV-GABA neurons). Notably, CCK- and PV-GABA neuronsdiffer substantially in electrophysiological and biochemicalfeatures (Freund and Katona, 2007; Armstrong and Soltesz,2012; Taniguchi, 2014). Briefly, CCK-GABA neurons exhibitmoderate, accommodating firing patterns (Cauli et al., 1997)and express serotonin type 3 (5-HT3) receptors (Morales andBloom, 1997) as well as cannabinoid type 1 (CB1) receptors(Katona et al., 1999). In contrast, PV-GABA neurons exhibit fast,non-accommodating firing rates (Cauli et al., 1997) and do notexpress either 5HT3 or CB1 receptors (Katona et al., 1999; Leeet al., 2010).

These contrasting features have stirred speculation that CCK-and PV-GABA neurons serve unique functions (Freund, 2003;Freund and Katona, 2007; Armstrong and Soltesz, 2012). PV-GABA neurons, which have been extensively studied, participatein gamma rhythm generation (Cardin et al., 2009; Sohal et al.,2009), neurogenesis (Song et al., 2013), sensory processing(Staiger et al., 1997, 2009; Moore and Wehr, 2013; Buetferinget al., 2014; Schneider et al., 2014; Siegle et al., 2014; Yekhlef et al.,2015), novelty recognition, associative learning and extinctionof learned behavior (Letzkus et al., 2011; Donato et al., 2013;Bissonette et al., 2014; Courtin et al., 2014; Wolff et al., 2014).In comparison to PV-GABA neurons, CCK-GABA neurons havebeen less scrutinized and their functions are less clear. It hasbeen argued that CCK-GABA neurons are ideally suited for the

regulation ofmood, anxiety and fear (Freund, 2003). Importantly,CCK-GABA neurons express 5HT3 and CB1 receptors, whichare involved in mood regulation (Park and Williams, 2012;McLaughlin et al., 2014). Further, CCK-GABA neurons targetpost-synaptic regions that are enriched with receptors involved inanxiety behaviors, namely CCK type B and α2 subunit-containingGABAA receptors (Nyiri et al., 2001; Raud et al., 2005; Koesteret al., 2013). An important role for CCK-GABA neurons inanxiety and fear has been shown, though this function hasbeen largely attributed to a modest population of neurons inthe amygdala (Truitt et al., 2009; Brown et al., 2014; Schmidtet al., 2014; Bowers and Ressler, 2015). The functions of otherpopulations of CCK-GABA neurons remain unclear.

Central to understanding the functions of CCK-GABAneurons is appreciating how these neurons are organizedwithin the central nervous system. Whereas PV-GABA neurondistribution has been extensively characterized using an arsenalof techniques (Celio, 1986; del Rio et al., 1994; Tamamaki et al.,2003), CCK-GABA neuron distribution has received surprisinglylittle attention. Initially, CCK-GABA neuron distribution wasinferred from antibody staining and autoradiography for CCKpeptide and its messenger RNA (Innis et al., 1979; Savasta et al.,1988; Ingram et al., 1989). Unfortunately, such techniques arenot selective for CCK-GABA neurons, as CCK is found in manyglutamatergic neurons (Hokfelt et al., 2002). More recently,CCK-GABA neuron distribution in several regions of thehippocampus and cortex has been assessed through co-stainingfor CCK peptide and the GABA synthesizing enzyme glutamicacid decarboxylase (GAD) (Hendry et al., 1984; Demeulemeesteret al., 1988; Kawaguchi and Kondo, 2002; Jinno and Kosaka,2003a,b). While highly informative, these studies investigateCCK-GABA neuron distribution only in select regions of thehippocampus and cortex. It is currently unknown how thedistribution of CCK-GABA neurons may vary in other regions.

The Cre recombinase-based genetic approach provides apowerful means to target a selective cell type using genepromoters with specific expression patterns, and may be of usein characterizing CCK-GABA neuron distribution. However, thespecificity of this approach is limited by the availability of Cretransgenic lines. Further, cell populations defined by the Credriver line alone are often heterogeneous. A recent study inCCK-Cre mice indicated that Cre activity is observed in bothGABAergic and glutamatergic cells (Taniguchi et al., 2011), likelybecause CCK is expressed in both cell populations (Hokfelt et al.,2002). Therefore, the Cre recombinase-based genetic approach byitself is inadequate for the specific study of CCK-GABA neurons.

In the present study, we applied a dual recombinase-basedgenetic strategy to label CCK- and PV-GABA cells in theforebrain. The dual recombinase-based strategy labels a highlyselective cell population which is defined by the overlappingexpression pattern of two signature genes that drive theexpression of Cre and Flpe recombinases. Specifically, Crerecombinase expression was driven by endogenous CCK or PVpromoters whereas Flpe recombinase expression was driven byDlx5/Dlx6 intergenic regulatory sequences that are specific toforebrain GABA neurons, including those cells derived from boththe medial and caudal ganglionic eminence (Miyoshi et al., 2010).

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Using a dual recombinase-responsive reporter allele, specificlabeling of CCK- and PV-GABA cells in the forebrain wasachieved by combining the Dlx5/6-Flpe allele with the CCK-Creand PV-Cre allele, respectively. In mice with all three alleles, wethen quantified the distribution of CCK- and PV-GABA cells ina wide volume of forebrain regions by unbiased automated cellcounting methods.

Materials and Methods

AnimalsTriple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FrePe mice(termed CCK-Frepe mice) and PV-Cre;Dlx5/6-Flpe;RC::FrePemice (termed PV-Frepe mice) were generated as follows:homozygous RC::FrePe mice (Bang et al., 2012; Robertsonet al., 2013) were crossed with Dlx5/Dlx6-FLPe mice [Tg(mI56i-FLPe)39Fsh/J, JAX#010815] to generate double transgenicDlx5/6-Flpe;RC::FrePemice, which were then crossed with eitherhomozygous CCK-ires-Cre mice [B6N.Cg-Ccktm1.1(cre)Zjh/J,JAX#019021] or PV-ires-Cre mice [B6;129P2-Pvalbtm1(cre)Arbr/J,JAX#008069]. Mice were group housed with ad libitum accessto food and water in a temperature-controlled room on a 12 hlight/dark cycle. Experimental procedures were in accordancewith the guidelines of the Canadian Council on Animal Care(CCAC) and the local Animal Care Committee at the Universityof Toronto.

ImmunohistochemistryTriple transgenic mice 3–8 months old were selected forexperiments. Mice were anesthetized with avertin andsubsequently underwent transcardial perfusion with 0.1 Mphosphate buffered saline (PBS; pH 7.4) followed by 4%paraformaldehyde (PFA) in PBS. Following perfusion, brainswere extracted and placed in 4% PFA at 4◦C for 24 h.Subsequently, brains were cryoprotected in a PBS solutioncontaining 30% sucrose at 4◦C for 48 h. Cryoprotected brainswere then cut into 40 μM sections using a cryostat (CM1520;Leica) held at −20◦C. From each brain, five sections wereobtained from the intermediate frontal cortex (Bregma = 1.72to 1.48 mm), intermediate parietal cortex (Bregma = −1.34to −1.94 mm) and rostral occipital cortex (Bregma = −2.46 to−3.28 mm) (15 sections total; Paxinos and Franklin, 2012).

For fluorescent immunohistochemistry in cell countingexperiments, free-floating tissue sections were rinsed with 0.1 MPBS and blocked with 5% normal donkey serum in 0.1%Triton-X-100 PBS (PBS-T) for 1 h at room temperature.Subsequently, sections were incubated with chicken polyclonalanti-green fluorescent protein (GFP; 1: 1000; ab13970; Abcam,Cambridge, MA, USA) and rabbit polyclonal anti-mCherry(1:1000; ab167453; Abcam) primary antibodies in PBS-Tfor 48 h at 4◦C. Thereafter, sections were rinsed withPBS-T and incubated with Alexa 488-conjugated donkey anti-chicken (1:1000; 703545145; Jackson ImmunoResearch; WestGrove, PA, USA) and Alexa 594-conjugated donkey anti-rabbit (1:1000; 715515152; Jackson ImmunoResearch) secondaryantibodies in PBS-T for 2 hr at room temperature. Sectionswere then rinsed with PBS-T and mounted on Superfrost

Plus slides (Fisher Scientific, Pittsburgh, PA, USA) andcoverslipped with Aquamount (Polysciences Inc., Warrington,PA, USA).

In experiments examining the colocalization of CCK orPV with GFP, separate sections were incubated with chickenpolyclonal anti-GFP antibody (1:1000; ab13970; Abcam)and either rabbit polyclonal anti-CCK-8 antibody in CCK-Frepe brain sections (1:1000; C2581; Sigma Aldrich; St.Louis, MO, USA) or mouse monoclonal anti-PV antibodyin PV-Frepe brain sections (1:1000; ab11427; Abcam) inPBS-T for 48 h at 4◦C. This was followed by incubationin PBS-T with Alexa 488-conjugated donkey anti-chickenantibody (1:1000; 703545145; Jackson ImmunoResearch) andeither Alexa 405-conjugated donkey anti-rabbit antibody(1:1000; ab175651; Abcam) in CCK-Frepe brain sections orAlexa 405-conjugated donkey anti-mouse antibody (1:1000;ab175658; Abcam) in PV-Frepe brain sections for 2 h at roomtemperature.

In experiments examining the colocalization of GFP ormCherry with GAD67, CCK- and PV-Frepe sections wereincubated with chicken polyclonal anti-GFP antibody (1:1000;ab13970; Abcam), goat polyclonal anti-mCherry antibody(1:1000; AB0040-20; Cedarlane, Burlington, ON, Canada) andmouse monoclonal anti-GAD67 antibody (1:1000; MAB5406;Billerica, MA, USA) in PBS-T for 48 h at 4◦C. Sectionswere then incubated in PBS-T with Alexa 488-conjugateddonkey anti-chicken antibody (1:1000; 703545145; JacksonImmunoResearch), Alexa 594-conjugated donkey anti-goatantibody (1:1000; 705515147; Jackson ImmunoResearch), andAlexa 405-conjugated donkey anti-mouse antibody (1:1000;ab175658; Abcam) for 2 h at room temperature.

Image Acquisition and Cell CountingFor cell counting experiments with wide field fluorescentmicroscopy, images of brain sections were generated using anFSX100 fluorescent microscope (Olympus). Alexa 488 and 594signals were captured using an U-MWIBA3 filter cube (Ex460-495, Em510-550, DM505) for Alexa 488 and an U-MWIG3filter cube (Ex530-550, Em575IF, DM570) for Alexa 594. Inacquired images, forebrain regions of interest (Table 1) weredelineated manually according to area definitions establishedby Paxinos and Franklin (2012). Automated cell counting ofGFP- and mCherry-labeled cells was performed using cellSens1.7 software (Olympus), which determined the total numberand numeric density of GFP- and mCherry-labeled cells indelineated brain regions. Cell density for each brain region wasexpressed in labeled cells/mm2. As 15 sections per animal wereanalyzed, multiple cell counts were available for every brainregion. For each animal, we calculated a single value per brainregion by averaging all values for that brain region across allsections.

For colocalization experiments, images were captured througha Quorum spinning disk confocal microscope (Zeiss) usinga 20x objective lens and were subsequently analyzed withVolocity Software (Perkin Elmer). Alexa Fluor 405, 488, and 594(secondary antibody signals) were excited with the 405, 491, and561 nm laser, respectively.

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TABLE 1 | Distribution of CCK- and PV-GABA cells within the hippocampus and cortex.

Density of GFP-labelledcells (cells/mm2)

% GFP-labelledcells/Total labelled cells

CCK-Frepe PV-Frepe CCK-Frepe PV-Frepe

(n = 7) (n = 4) (n = 7) (n = 4)

Region Subregion Full name Mean SEM Mean SEM Mean SEM Mean SEM D score

Hippocampus dCA1 CA1, dorsal 61.6 7.5 35.5 4.8 31.7 3.0 16.4 3.2 15.3

dCA3 CA3, dorsal 51.7 6.2 50.3 11.3 25.7 3.1 23.3 6.2 2.4

dDG Dentate gyrus, dorsal 20.5 2.8 19.6 1.3 16.1 2.2 12.8 2.0 3.3

dSub Subiculum, dorsal 42.1 3.4 108.1 10.1 22.7 2.6 37.0 4.8 −14.3

vCA1 CA1, ventral 92.4 15.4 43.2 3.9 29.0 3.8 14.6 3.1 14.4

vCA3 CA3, ventral 81.9 12.6 72.6 19.0 25.4 3.8 20.4 6.5 5.0

vDG Dentate gyrus, ventral 30.1 4.6 6.6 0.6 9.5 1.4 2.7 0.9 6.8

Frontal Cg Cingulate cortex 63.9 7.3 87.9 5.5 21.6 1.9 23.1 1.5 −1.5

DP Dorsal peducunlar region 73.3 11.8 41.3 7.4 24.7 3.2 10.4 0.8 14.2

IL Infralimbic cortex 75.4 9.7 34.9 2.6 21.4 2.5 7.5 0.6 13.9

M1 Motor cortex, Primary 56.4 8.1 109.1 24.7 19.7 2.5 31.6 1.7 −11.8

M2 Motor cortex, Secondary 70.2 7.1 100.2 13.2 24.1 2.9 30.1 1.3 −6.1

PL Prelimbic cortex 69.9 6.9 49.1 7.0 19.7 1.7 10.7 1.5 8.9

Retrosplenial Rsd Retrosplenial cortex, dysgranular 40.9 6.3 193.8 27.8 16.1 2.9 43.9 3.8 −27.8

RsgA Retrosplenial cortex, granular region A 42.4 7.4 95.3 17.0 21.4 4.7 27.6 4.3 −6.2

RsgB Retrosplenial cortex, granular region B 31.0 4.2 137.9 34.0 13.5 2.0 31.1 5.6 −17.6

RsgC Retrosplenial cortex, granular region C 33.7 6.6 205.4 43.3 14.1 3.1 44.5 5.2 −30.5

Parietal PtaL Parietal Association Area, Lateral 71.1 4.6 166.1 20.5 20.3 2.5 35.0 3.2 −14.7

PtaM Parietal Association Area, Medial 60.5 4.3 161.9 17.5 20.7 3.2 33.5 2.3 −12.8

PtaP Parietal Association Area, Posterior 49.6 8.3 155.3 18.2 15.1 2.7 36.8 4.1 −21.7

S1 Somatosensory Area, Primary 39.9 6.4 148.7 19.1 13.3 2.4 39.3 6.6 −26.0

S1bf Somatosensory Area, Primary (barrel) 40.7 6.1 177.3 29.3 12.5 2.1 42.5 5.4 −30.1

S1tr Somatosensory Area, Primary (trunk) 63.5 8.9 175.2 26.3 17.6 2.9 40.3 5.1 −22.7

S1ulp Somatosensory Area, Primary (upper-lip) 33.8 6.7 146.1 11.6 11.7 2.4 39.4 3.6 −27.7

S2 Somatosensory Area, Secondary 38.5 5.2 128.2 3.8 13.4 2.1 35.0 2.2 −21.6

Occipital V1b Visual cortex, Primary Basal 24.1 3.7 178.8 22.1 7.5 1.2 41.6 5.7 −34.0

V1m Visual cortex, Primary Medial 29.5 6.0 173.3 18.1 8.7 1.7 39.1 5.5 −30.4

V2l Visual cortex, Secondary Lateral 45.1 8.8 164.4 13.2 13.8 2.6 37.3 3.9 −23.5

V2ml Visual cortex, Secondary Mediolateral 55.2 9.6 136.1 12.0 15.1 2.7 30.9 4.4 −15.8

V2mm Visual cortex, Secondary Mediomedial 57.8 7.7 101.9 9.8 17.3 2.4 23.6 1.6 −6.3

Temporal Aud1 Auditory cortex, Primary 28.3 4.0 147.6 10.0 9.8 1.6 36.7 2.6 −26.8

AudD Auditory cortex, Dorsal 44.5 6.4 139.2 9.1 14.1 2.4 36.1 3.7 −21.9

AudV Auditory cortex, Ventral 38.9 4.6 109.4 8.1 14.1 2.0 28.7 2.4 −14.7

Ect Ectorhinal cortex 34.6 6.2 21.6 2.0 17.8 2.9 13.9 1.0 4.0

EntDI Entorhinal cotex, Dorsointermediate 32.1 4.2 18.4 4.1 15.3 3.0 8.9 2.3 6.4

EntDL Entorhinal cortex, Dorsolateral 50.2 7.8 46.5 3.2 17.1 2.6 8.2 2.0 8.9

PRh Perirhinal cortex 43.7 5.2 43.0 3.2 19.6 2.5 15.2 1.5 4.4

Tea Temporal association area 53.3 8.2 58.1 7.4 18.1 2.8 15.0 1.4 3.1

In each brain region, the density of CCK- and PV-GABA cells (units/mm2 ) and their relative contribution to the GABA cell population (%) is shown. Also included below isthe ‘balance’ between CCK- and PV-GABA cells in a given brain region (D), expressed as the subtractive difference between CCK-GABA and PV-GABA cell percentage.

In experiments examining the colocalization of CCK orPV with GFP, four sections per animal were counted from a725μm2 area in the CA1 subfield of the hippocampus. Cells werecounted in the stratum radiatum in CCK-Frepe brain sectionsand the stratum pyramidale from PV-Frepe brain sections, asthese areas show the highest density of CCK- and PV-GABAcells in the hippocampus, respectively. The specificity of our

dual-recombinase approach was assessed by determining thepercentage of GFP-labeled cells that were immunopositive forCCK (in CCK-Frepemice) or PV (in PV-Frepemice).

Similarly, in experiments examining the colocalization of GFPormCherry with GAD67, sections were counted fromCCK-Frepe(n = 2) and PV-Frepemice (n = 2) in the stratum radiatum. Wedetermined the percentage of fluorescently labeled cells (either

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GFP or mCherry) that were immunopositive for GAD67, and thepercentage of GAD67-labeled cells also positive for either GFP ormCherry.

Statistical AnalysisAll analysis was performed using GraphPad Prism 6.0 forWindows. Separate two-way analysis of variance (ANOVA) testswere performed for structures belonging to the hippocampus,frontal cortex, retrosplenial cortex, parietal cortex, occipitalcortex and temporal cortex. In each two-way ANOVA, mousegenotype was used as a between-subjects factor (either CCK-Frepe or PV-Frepe) while brain region was used as a within-subjects factor. Post-hoc analysis was performed using Sidak’smultiple comparison test with the level of significance set atp = 0.05. When analyzing the percentage of GFP-labeled cells ineach layer of the hippocampus, separate one-way ANOVA testswere performed for each subfield (CA1, CA3, and DG).

Results

Intersectional Genetic Labeling of CCK-GABAand PV-GABA CellsTo selectively label CCK- and PV-GABA cells for counting,we employed a dual recombinase-based intersectional geneticstrategy (Jensen and Dymecki, 2014) (Figure 1). We firstcrossed the Dlx5/6-Flpe mouse line, which provides a selectivegenetic access to forebrain GABAergic neurons, with a dualrecombinase-responsive reporter line, RC::FrePe (Engleka et al.,2012) (Figure 1A). Subsequently, double transgenic Dlx5/6-Flpe;RC::FrePe mice were crossed with either CCK-Cre or PV-Cre mouse lines, in which the expression of Cre recombinaseis restricted to CCK and PV neurons, respectively. Theresulting triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FrePe mice(CCK-Frepe, n = 7) and PV-Cre;Dlx5/6-Flpe;RC::FrePe mice(PV-Frepe, n = 4) were used in cell counting experiments(Figure 1B). In CCK- and PV-Frepe mice, cells expressingFlpe and Cre recombinase (i.e., CCK- or PV-GABA cells) arelabeled with reporter enhanced green fluorescent protein (GFP)(Figure 1B). In contrast, cells expressing only Flpe recombinase(i.e., nonCCK-GABA and nonPV-GABA cells) are labeled withthe reporter protein mCherry. Cells expressing Cre recombinasealone, or neither recombinase, are unlabeled.

Prior to cell counting, we examined the specificity of labelingin the intersectional genetic approach. In CCK- and PV-Frepemice, GFP reporter expression was compared with CCK and PVimmunofluorescence, respectively (Figures 1C,D). In sectionsfrom CCK-Frepe mice (n = 4), 85.0% of GFP-expressing cellswere positive for CCK immunoreactivity. In sections from PV-Frepe mice (n = 4), 87.1% of GFP-expressing cells were positivefor PV immunoreactivity. These data indicate that the largemajority of CCK- and PV-GABA cells are labeled with GFP inCCK- and PV-Frepemice, and verify a high specificity of labelingusing the intersectional approach.

Furthermore, we examined the efficacy of the intersectionalgenetic approach for labeling GABA cells. Previous studieshave shown that the Dlx5/6-Flpe line can be used to label

the majority of GABA cells derived from the medial, caudaland lateral ganglionic eminences (Miyoshi et al., 2010). Onestudy reported that as many as ∼90% of GABA cells in theneocortex may be labeled by combining the Dlx5/6-Flpe linewith a rosa26 knock-in Flpe reporter line (Imayoshi et al., 2012).To determine the efficacy of GABA cell labeling, we quantifiedthe percentage of fluorescently labeled cells (either with GFP ormCherry) that were immunopositive for the enzyme glutamicacid decarboxylase 67 (GAD67), which is a marker of GABAneurons (Figure 1E). In a sample of CCK-Frepe (n = 2) and PV-Frepe brains (n = 2), we observed that 71.5% of fluorescentlylabeled cells in the hippocampus were also positive for GAD67.Additionally, we observed that 70.0% of GAD67-positive cellswere also positive either GFP or mCherry. These data verify thatthe majority of GABA cells are labeled using the intersectionalapproach.

HippocampusTo further validate the effectiveness of the intersectional geneticapproach, we quantified GFP-labeled cells in the hippocampusof CCK- and PV-Frepe mice. We reasoned that this structurewould provide an ideal reference point as it is populated by manyGABAergic neurons, including CCK- and PV-GABA neurons,of well-characterized distribution (Kosaka et al., 1985, 1987;Nomura et al., 1997; Jinno and Kosaka, 2006). Additionally, thehippocampus is a convenient target for histological examinationas its circuitry and anatomy have been extensively studied(Freund and Buzsaki, 1996; Strange et al., 2014). Thus,we tabulated the percentage of GFP-labeled cells within thehippocampus of CCK- and PV-Frepe mice, and compared thesevalues to those established for CCK- and PV-GABA cells usingother labeling techniques.

In CCK- and PV-Frepe mice, cells labeled with GFPor mCherry were quantified using unbiased automated cellcounting. The percentage of CCK- and PV-GABA cells wasdefined as the percentage of fluorescently labeled cells that wereGFP-labeled cells (i.e., GFP cells/[GFP cells + mCherry cells]).In our analysis of the hippocampus, we counted labeled cells inthe Cornu Ammonis subfields (CA1, CA3), dentate gyrus (DG)and subiculum (Sub). As the dorsal and ventral hippocampus areoften considered anatomically and functionally distinct (Nomuraet al., 1997; Strange et al., 2014), dorsal and ventral subregionswere defined separately by using the rhinal fissure as a landmark.Subregions above the rhinal fissure were considered to belongto the dorsal hippocampus whereas subregions below wereconsidered to belong to the ventral hippocampus. In total,eight hippocampal subregions were counted (dCA1, dCA3, dDG,dSub, vCA1, vCA3, vDG, and vSub) (Table 1, Figure 2). As oursampling method focused on the intermediate sections of themouse brain, the caudal vSub was often absent. Accordingly, thisregion was excluded from analysis.

The density and percentage of GFP-labeled cells withinhippocampal subregions of CCK- and PV-Frepemice is shown inTable 1. Two-way ANOVA detected a significant interaction ofgenotype × brain region on the percentage of GFP-labeled cells[F(6,62) = 3.72, p < 0.01]. In both the dCA1 and vCA1 subfields,the percentage of CCK-GABA cells was greater than PV-GABA

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FIGURE 1 | Selective labeling of CCK- and PV-GABA cells using the dual recombinase–based intersectional genetic strategy. (A) Top. A dualrecombinase-responsive reporter allele, RC::FrePe, contains two transcriptional stop cassettes. The first stop cassette is flanked by vertically oriented FRT sites(rectangles, denoted with F ) and the other by directly oriented loxP sites (triangles, denoted with P). The loxP-flanked stop cassette also contains mCherry-encodingsequences. Middle: Flpe-mediated stop cassette removal results in mCherry expression. The remaining loxP-flanked stop cassette prevents GFP expression.Bottom: Upon removal of both stop cassettes, requiring Flpe- and Cre-mediated excisions, expression of GFP is turned on and expression of mCherry is turned off.The RC::FrePe allele is knocked-in to the Gt(ROSA)26Sor (R26) locus with CAG (chicken β-actin and CMV enhancer) promoter elements. (B) Venn diagramsillustrating intersectional and subtractive cell populations labeled by the intersectional approach using the RC::FrePe allele. The Dlx5/6-Flpe allele is specific toGABAergic cells in the forebrain. In triple transgenic mice inheriting all three alleles (CCK-Cre;Dlx5/6-Flpe;RC::FrePe mice or PV-Cre;Dlx5/6-Flpe;RC::FrePe mice),cells expressing both Cre and Flpe alleles (i.e., intersectional population) represent CCK- or PV-GABA cells, and are labeled with GFP. In contrast, cells expressingonly Flpe (i.e., subtractive population) represent nonCCK-GABA or nonPV-GABA neurons and are labeled with mCherry. (C,D) Specificity of labeling GABA cellsusing the intersectional genetic strategy. (C) Confocal images of the CA1 stratum radiatum in CCK-Frepe mice (top) and the CA1 stratum pyramidale in PV-Frepemice (bottom). GFP-expressing cells are labeled in green whereas CCK+ cells (top) and PV+ cells (bottom) are shown in blue. The border between the stratumpyramidale and stratum radiatum is denoted by the dotted line. Scale bar = 20 μm. (D) Percentage of GFP-expressing cells in CCK- and PV-Frepe mice that areCCK+ (orange) or PV+ (blue), respectively. (E) Efficacy of GABA cell labeling in the intersectional genetic approach. Confocal image shows the CA1 stratum radiatumin PV-Frepe mice. GFP- and mCherry-expressing cells are shown in green and red, respectively, whereas GAD67+ cells are shown in blue. Scale bar = 20 μm.

cells (p < 0.05, Figures 2A,B). The relative abundance of CCK-GABA cells within the ventral hippocampus of CCK-Frepe miceis consistent with past reports, which show a relatively highdensity of CCK-GABA cells in this region (Jinno and Kosaka,

2003b, 2006; Meyer, 2014). Further, we observed that PV-GABAcells were more numerous in the dSub (p < 0.05, Figures 2A,B),as shown previously (Pitkanen and Amaral, 1993; Boccara et al.,2014). In the remaining subregions of the hippocampus (dCA3,

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FIGURE 2 | Distribution of CCK- and PV-GABA cells within thehippocampus. (A) Hippocampal sections from CCK-Frepe (left) andPV-Frepe mice (right). GFP-labeled cells represent CCK- and PV-GABA cells,respectively. (B) Percentage contribution of CCK-GABA cells (orange) andPV-GABA cells (blue) to the GABA neuron population in each hippocampalsubregion. CCK-GABA cells were comparatively more abundant in the dCA1and vCA1 regions whereas PV-GABA cells were more abundant in the dSubregion. (C) The total density of GABA cells, defined as the sum of GFP- andmCherry-labeled cell density, did not differ between CCK- and PV-Frepe mice.(D,E) Distribution of CCK- and PV-GABA cells by hippocampal layer.CCK-GABA cells were most common in the sr layer but were also found in thesp and so layers. PV-GABA cells were more concentrated in the sp layer, butwere also found in the so layer. Abbreviations by subregion: dCA1, dorsalCA1, dCA3, dorsal CA3, dDG, dorsal dentate gyrus, dSub, dorsal subiculum,vCA1, ventral CA1, vCA3, ventral CA3 and vDG, ventral dentate gyrus.Abbreviations by layer: hi, hilus, sg, stratum granulosum, sl, stratum lucidum,slm, stratum lacunosum-moleculare, sm, stratum moleculare, so, stratumoriens, sp, stratum pyramidale and sr, stratum radiatum. Significance at thep < 0.05 level is denoted with an asterisk. Scale bar = 500 μM.

vCA3, dDG, and vDG), the percentages of CCK- and PV-GABAcells did not differ (all ps > 0.05, Figures 2A,B). The observeddifferences in the percentage of CCK- and PV-GABA cells acrosshippocampal subregions were not due to differences in totalGABA cell count within the hippocampus, as the total density oflabeled cells (GFP + mCherry) did not differ between CCK- andPV-Frepemice by subregion (p > 0.05, Figure 2C). Additionally,

these counts were not due to an experimenter’s bias in thedelineation of hippocampal subregions. When hippocampal cellcounts were re-analyzed by an experimenter blind to condition,total GABA cell count did not differ in any subregion betweenCCK- and PV-Frepemice (all ps > 0.05, data not shown).

Collectively, these data show that the distribution of GFP-labeled cells in CCK- and PV-Frepe mice is consistent withthe known distribution of CCK- and PV-GABA cells withinthe subregions of the hippocampus (Kosaka et al., 1985, 1987;Nomura et al., 1997; Jinno and Kosaka, 2006). However, thedistribution of GABAergic cells also differs by hippocampal celllayer as well as by subregion (Kosaka et al., 1985; Schiffmann andVanderhaeghen, 1991; Freund and Buzsaki, 1996; Botcher et al.,2014). To examine the layer-specific patterns of distribution ofCCK- and PV-GABA cells, we counted GFP-labeled cells withinthe layers of the hippocampus of CCK- and PV-Frepe mice.In a sample of dorsal hippocampal sections, GFP-labeled cellswere quantified in the stratum oriens (CA1so, CA3so), stratumpyramidale (CA1sp, CA3sp), stratum lacunosum-moleculare(CA1slm, CA3slm), stratum lucidum (CA3sl), stratum moleculare(DGsm), stratum granulosum (DGsg) and hilar region (DGhi)(Figures 2D,E).

One-way ANOVA detected a significant effect of hippocampallayer on the percentage of GFP-labeled cells in the CA1 [CCK-Frepe: F(3,24) = 145.4, p < 0.0001; PV-Frepe: F(3,12) = 64.71,p < 0.0001], CA3 [CCK-Frepe: F(4,30) = 77.29, p < 0.0001;PV-Frepe: F(4,15) = 125.7, p < 0.0001] and DG subfields [CCK-Frepe: F(2,18) = 21.35, p < 0.0001; PV-Frepe: F(2,9) = 12.97,p < 0.01]. In the CA1 subfield, CCK-GABA cells were commonlyfound in the CA1so, CA1sp and CA1sr layers but not in theCA1slm layer (all ps < 0.05). The content of CCK-GABAcells was greatest in the CA1sr layer but was also high in theCA1sp layer, as shown previously (Jinno and Kosaka, 2006)(Figures 2D,E). A similar distribution of CCK-GABA cells wasseen in the CA3 subfield, with GFP-labeled cells being morenumerous in the CA3sr layer (all ps < 0.05, Figures 2D,E). Incontrast, PV-GABA cells were highly concentrated in the sp layerof CA1 and CA3 (all ps < 0.05, Figures 2D,E), as shown byothers (Jinno and Kosaka, 2002). In the DG, both CCK- and PV-GABA cells were numerous in the DGsg layer and infrequent inthe DGml, as demonstrated previously (Jinno and Kosaka, 2002,2003a,b). Overall, these data demonstrate that the pattern of GFP-labeling in the Frepe model is highly consistent with that shownby antibody labeling studies.

Frontal CortexNext, GFP-labeled cells were quantified in the frontal cortex ofCCK- and PV-Frepemice. In this region, CCK is considered oneof the dominant neuropeptides (Hornung et al., 1992) and manycells are positive for CCK messenger RNA (Savasta et al., 1988;Oeth and Lewis, 1993). While these findings might imply a largepopulation of CCK-GABA neurons is present, early studies usingantibody labeling suggested that such cells represented only asmall proportion of GABAergic neurons (1–5%; Kubota et al.,1994; Kubota and Kawaguchi, 1997). In contrast, more recentresearch using single-cell reverse transcription-polymerase chainreaction (scRT-PCR) techniques suggests that many interneurons

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in the cortex express CCK mRNA (∼30–40%; Gallopin et al.,2006), though it is debated whether transcript expression reflectsCCK-GABA neuron identity (Tricoire et al., 2011). Theseconflicting results have generated some controversy regardingthe abundance and importance of CCK-GABA neurons in thefrontal cortex. We reasoned that the intersectional approach,which labels a subpopulation of GABAneurons according to theirgenetic history for CCK expression, would provide insight intothis situation.

Within the frontal cortex, GFP-labeled cells were countedin the cingulate cortex (Cg), dorsal peduncular region (DP),infralimbic (IL) cortex, primary motor cortex (M1), secondarymotor cortex (M2) and prelimbic (PL) cortex (Figure 3, Table 1).These particular subregions of the frontal cortex are relativelyeasy to distinguish, contain elaborate networks of CCK- andPV-GABA neurons, and are implicated in the pathogenesis ofneurodegenerative and neuropsychiatric diseases (Bachus et al.,1997; Penschuck et al., 2006; Abdul-Monim et al., 2007; Braunet al., 2007; Falco et al., 2014; Uchida et al., 2014; Wischhof et al.,2015). Two-way ANOVA detected a significant interaction ofgenotype × brain region on the percentage of GFP-labeled cells[F(5,48) = 11.27, p < 0.001]. Within the IL and DP regions ofthe frontal cortex, CCK-GABA cells were more numerous thanPV-GABA cells (ps < 0.05; Figure 3). In the PL, CCK-GABAcells also tended to be more numerous, though the difference wasnot significant. In contrast to the relative abundance of CCK-GABA cells in medial prefrontal cortex subregions (IL/PL/Cg),PV-GABA cells were significantly more abundant in the M1subregion (ps < 0.05, Figure 3) and tended to be more abundantin M2 subregion. Collectively, PV-GABA cells constituted ∼40%of all GABAergic cells inM1 andM2, as shown previously (Condeet al., 1994; Tamamaki et al., 2003; Uchida et al., 2014). Therewere no differences in the percentages of CCK- and PV-GABAcells in the Cg (p > 0.05). In summary, these data suggest thatCCK-GABA cells comprise a larger proportion of the GABAergicpopulation in the medial prefrontal cortex than in the adjacentmotor cortex.

Retrosplenial CortexThe retrosplenial cortex plays an important role in visuospatialprocessing, memory and extinction (Vann et al., 2009; Czajkowskiet al., 2014; Kwapis et al., 2014; Nelson et al., 2015; Todd et al.,2015) but has received surprisingly little anatomical investigation,with only limited information suggesting abundant PV-GABAneurons (del Rio et al., 1994). As the circuitry of the retrosplenialcortex is still being unraveled (Vann et al., 2009), the contributionof other GABAergic neurons, including CCK-GABA neurons,needs to be characterized.

GFP-labeled cells were counted in the subdivisions ofthe retrosplenial cortex, including the dysgranular (Rsd) andgranular regions (RsgA, RsgB and RsgC) (Table 1). Two-wayANOVA detected a significant interaction of genotype × brainregion on the percentage of GFP-labeled cells [F(3,36) = 3.77,p < 0.05]. PV-GABA cells were significantly more numerousthan CCK-GABA cells in the Rsd, RsgB, and RsgC subdivisions(p < 0.05). Additionally, PV-GABA cells tended to be morenumerous in the RsgA subdivision but the difference was

FIGURE 3 | CCK-GABA cells are most numerous within the medialprefrontal cortex. (Top) Sections of the prefrontal cortex in CCK- andPV-Frepe mice. (Bottom) Percentage contribution of CCK- and PV-GABAcells to the total GABA neuron population by subregion of the frontal cortex. Inthe DP and IL regions, CCK-GABA cells were more abundant than PV-GABAcells. CCK-GABA cells also tended to be more abundant in the PL region.PV-GABA cells were more abundant in M1 region and tended to be moreabundant in the M2 region. Abbreviations: Cg, cingulate cortex, DP, dorsalpeduncular region, IL, infralimbic cortex, M1, primary motor cortex, M2,secondary motor cortex, PL, prelimbic cortex. Significance at the p < 0.05level is denoted with an asterisk. Scale bar = 500 μM.

not significant. These data strongly suggest that PV-GABAneurons are by far the dominant perisomatic interneurons in theretrosplenial cortex.

Parietal CortexThe parietal cortex comprises a large portion of the mousebrain and contains dense networks of perisomatic interneurons(Hendry et al., 1983; Houser et al., 1983). PV-GABA neuronsare especially numerous in the parietal cortex (Celio, 1986;Ren et al., 1992; del Rio et al., 1994; Powell et al., 2003; Xuet al., 2010; Perrenoud et al., 2013), where they regulate manyfunctions, including the processing of sensory information fromthe thalamus regarding tactile perception (Staiger et al., 1997,2009; Siegle et al., 2014). As antibody labeling studies haveestablished that PV-GABA neurons are more abundant thanmost other GABA neurons in the parietal cortex, we expectedPV-GABA cells to be highly numerous in the Frepemodel.

Due to the substantial volume of the parietal cortex, weelected to sample intermediate subregions only. Specifically,GFP-labeled cells were counted in several subdivisions of the

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primary somatosensory cortex (S1bf, S1tr, S1ulp, S1), parietalassociation cortex (PtaL, PtaM, and PtaP) and in the secondarysomatosensory cortex (S2) (Table 1, Figure 4). Two-way ANOVAdetected a main effect of genotype on the percentage of GFP-labeled cells in the parietal cortex [F(1,71) = 179.3, p < 0.0001],but not an interaction of genotype × brain region. In allregions of the parietal cortex but the PtaM, PV-GABA cellswere significantly more numerous than CCK-GABA cells (allps < 0.05, Figure 4). These data support the notion that PV-GABA cells are the most abundant perisomatic interneurons inthe parietal cortex (Lee et al., 2010).

Occipital CortexSimilar to the parietal cortex, the occipital cortex is thought toinclude large quantities of PV-GABA neurons (∼40%) and lowquantities of other GABAergic neurons (Demeulemeester et al.,1988; Hornung et al., 1992; Beaulieu, 1993; del Rio et al., 1994;Gonchar et al., 2007; Xu et al., 2010). However, most prior studiesof the occipital cortex have focused on the primary visual cortex(V1) (Hornung et al., 1992; Beaulieu, 1993; del Rio et al., 1994)and have not extensively studied GABAergic cell content in othersubregions, such as the secondary visual cortex (V2). In theseareas, the relative distribution of GABAergic neurons is less clear.To determine whether the distribution of CCK- and PV-GABAcells varied across the occipital cortex, GFP-labeled cells weremeasured in the main subdivisions of the primary visual cortex

(V1b, V1m) and secondary visual cortex (V2mm, V2ml, and V2l)(Figure 5).

Two-way ANOVA detected a significant interaction ofgenotype × brain region on the percentage of GFP-labeled cells[F(4,45) = 6.51, p < 0.01]. In all subdivisions of the occipitalcortex but V2mm, PV-GABA cells were more numerous thanCCK-GABA cells (all ps < 0.05, Figure 5). In the V2mmsubregion, PV-GABA cells tended to be more abundant, butthe difference was not significant according to post hoc analysis(p > 0.05). Interestingly, the observation that PV-GABA cells areless numerous in the V2mm area than in the surrounding visualcortices has also been made in a previous antibody labeling studyfor PV (del Rio et al., 1994). Overall, there was a prominent trendobserved wherein CCK-GABA cells tended to be more numerousin secondary visual cortex regions (V2mm, V2ml, V2l) than inthe primary visual cortex regions (V1b, V1m).

Temporal Cortex and Association AreasThe temporal cortex is critical for auditory processing,multimodal associations and memory formation (Squireet al., 2004; Schreiner and Winer, 2007). This region containslarge networks of GABAergic basket cells, many of which arethought to be PV-GABA neurons (Blazquez-Llorca et al., 2010).PV-GABA neuron networks of the temporal cortex have beenextensively described (Celio, 1986; Pitkanen and Amaral, 1993;McMullen et al., 1994) and regulate many functions, including

FIGURE 4 | Abundance of PV-GABA cells in the parietal cortex. (Top) Sections of the intermediate parietal cortex from CCK- and PV-Frepe mice. (Bottom)Relative contribution of CCK- and PV-GABA cells to the total GABA neuron population by subregion of the parietal cortex. PV-GABA cells were comparatively morenumerous in every subregion but the PtaM. Abbreviations: PtaL, lateral parietal association area, PtaM, medial parietal association area, PtaP, posterior parietalassociation area, S1, primary somatosensory area, S1bf, somatosensory area (barrel), S1tr, somatosensory area (trunk), S1ulp, somatosensory area (upper lipregion), S2, secondary somatosensory area. Significance at the p < 0.05 level is denoted with an asterisk. Scale bar = 500 μM.

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FIGURE 5 | Abundance of PV-GABA cells in the occipital cortex. (Top)Sections of the occipital cortex from CCK- and PV-Frepe mice. (Bottom)Percentage contribution of CCK- and PV-GABA cells to the total GABA cellpopulation by subregion of the occipital cortex. PV-GABA cells arecomparatively more numerous in every subregion but the V2mm, where theytended to be more numerous. Abbreviations: V1b, primary visual cortex, basalregion; V1m, primary visual cortex, medial region; V2l, secondary visualcortex, lateral region; V2ml, secondary visual cortex, mediolateral region;V2mm, secondary visual cortex, mediomedial region. Significance at thep < 0.05 level is denoted with an asterisk. Scale bar = 500 μM.

the processing of auditory information and spatial mapping(Moore and Wehr, 2013; Buetfering et al., 2014; Schneideret al., 2014; Yekhlef et al., 2015). Comparatively, there is littleanatomical or functional understanding of CCK-GABA neuronnetworks within the temporal cortex. While antibody labelingstudies suggest that many CCK-expressing neurons are indeedpresent, particularly within the ventrolateral temporal region(Innis et al., 1979; Miceli et al., 1987; Ingram et al., 1989),it is unknown whether these cells are CCK-GABA neuronsspecifically.

GFP-labeled cells in CCK- and PV-Frepe mice wereenumerated in the intermediate regions of the temporalcortex. In our sampling method, we included subdivisionsof the auditory cortex (Aud1, AudD, AudV) as well as thetemporal association cortex (Tea), ectorhinal cortex (Ect),perirhinal cortex (Prh) and entorhinal cortex (EntDI andEntDL) (Table 1, Figure 6). Two-way ANOVA detected agenotype × brain region interaction on the percentage of GFP-labeled cells [F(7,71) = 14.45, p < 0.0001]. In all subdivisions ofthe auditory cortex, PV-GABA cells were highly abundant andmore numerous than CCK-GABA cells (all ps < 0.05, Figure 6).PV-GABA cell density was much lower outside of the auditorycortex, as observed in other species (McMullen et al., 1994).Interestingly, the transition from the ventral auditory cortex(AudV) into the area of the rhinal fissure (Tea, Ect, and PRh)coincided with an abrupt reduction in PV-GABA cell percentage.Surprisingly, the reverse trend in distribution was observed for

FIGURE 6 | Contrasting distribution of CCK- and PV-GABA cells in thetemporal cortex. (Top) Sections of the temporal cortex from CCK- andPV-Frepe mice. (Bottom) Percentage contribution of CCK- and PV-GABAcells to the total GABA cell population by subregion of the temporal cortex.PV-GABA cells are significantly more numerous in auditory cortex regions.Ventral to the rhinal fissure, PV-GABA cells become less common andCCK-GABA cells become more numerous. Abbreviations: V1b, primary visualcortex, basal region; V1m, primary visual cortex, medial region; V2l, secondaryvisual cortex, lateral region; V2ml, secondary visual cortex, mediolateralregion; V2mm, secondary visual cortex, mediomedial region. Significance atthe p < 0.05 level is denoted with an asterisk. Scale bar = 500 μM.

CCK-GABA cells, which tended to be more numerous in theTea, Ect, and PRh areas than in auditory cortex. As a result ofthese contrasting trends, the percentage count of CCK- andPV-GABA cells was comparable in temporal association areas. Infact, CCK-GABA cell percentage even tended to be higher thanPV-GABA cell percentage in the examined subdivisions such asthe entorhinal cortex (EntDI, EntDL), though the difference wasnot significant.

Comparative Distribution of CCK- andPV-GABA CellsAs CCK- and PV-GABA neurons are thought to regulatepyramidal cell activity by distinct mechanisms (Freund, 2003;Freund and Katona, 2007), they may be specialized for certaincortical network activities. Within the cortex, informationprocessing is known to differ substantially between the primaryand secondary regions. Primary cortical regions (such as Aud1,

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V1, S1, and M1) are typically situated early in hierarchalnetworks (Van Essen et al., 1992; Kaas et al., 1999; Kimet al., 2014b), and must rapidly transmit information to otherbrain regions for higher-order processing. In comparison,secondary/association cortical regions (AudD/AudV, V2, S2and M2) participate in later stages of hierarchical informationprocessing, and likely integrate many inputs over a longertimeframe. We reasoned that these differences in processingneeds may be reflected in a differential balance of perisomaticinterneurons. To see if the relative content of perisomaticinterneurons differs between primary and secondary areas ofthe cortex, we calculated the subtractive difference betweenthe average CCK-GABA cell percentage and the average PV-GABA cell percentage (termed D score) in all brain regionsstudied (Figure 7). Notably, this analysis is distinct from thoseconducted previously, as it directly compares CCK- and PV-GABA cells to each other and does not include other types ofGABA cells.

Graphing the D score by rank revealed several interestingtrends of CCK-GABA cell distribution. The D score of anysecondary/association cortical area (green bars) was always higherthan the D score of the corresponding primary cortical area(red bars). This trend was observed in the case of the occipitalcortex (V2mm/V2ml/V2L versus V1b/V1m), auditory cortex(AudD/AudV versus Aud1), sensory cortex (PtaL/PtaM/S2versus S1bf/S1tr/S1ulp) and motor cortex (M2 versus M1).This finding suggests that CCK-GABA cells tend to comprisea greater proportion of the perisomatic interneuron populationin secondary/association cortex than in the primary cortex.In contrast, PV-GABA cells tend to comprise a greaterproportion of the GABA cells in the primary cortex, a resultconsistent with prior antibody labeling studies (Cruikshank et al.,2001).

Discussion

To our knowledge, the current study represents the firstcomprehensive, systematic analysis of CCK- and PV-GABAinterneuron distribution in the forebrain. Using a dualrecombinase-based genetic strategy, we quantified two majorpopulations of perisomatic interneurons in a wide volume ofdiscrete brain regions. This extensive sampling contrasts withthe focus of previous studies, which examined fewer areas(Hendry et al., 1984; Celio, 1986; Demeulemeester et al., 1988;del Rio et al., 1994; Kawaguchi and Kondo, 2002; Jinno andKosaka, 2003a,b; Tamamaki et al., 2003). By broadly quantifyingthese cells, we identified novel trends in their distribution.CCK-GABA cells were shown to be remarkably abundant inseveral brain regions outside of the hippocampus, includingthe medial prefrontal cortex and ventrolateral temporal cortex.Additionally, CCK-GABA cells tended to be more abundantin secondary/association areas of the cortex than in primarysensory areas.

Overall, the distribution of CCK- and PV-GABA cells detectedin the current study was consistent with that demonstrated byantibody labeling studies. While the percentage of PV-GABAcells was in agreement with past reports, the percentage ofCCK-GABA cells in our study was higher. There are twoexplanations for this discrepancy. The first possibility is thatprevious studies may underestimate the number of CCK-GABAneurons due to a low efficacy of antibody labeling for CCK,as has been suggested by Gallopin et al. (2006) and impliedby the results of Xu et al. (2010). In contrast to antibodylabeling, the expression of GFP reporter in the CCK-Frepemouse is highly sensitive to CCK transcript expression andreadily occurs even in CCK-GABA neurons containing a verysmall amount of CCK peptide. An alternative possibility as

FIGURE 7 | Perisomatic interneuron balance and its relationship to cortical function. Cell balance was estimated using the delta score (D), which was thesubtractive difference between CCK-GABA and PV-GABA cell percentage. Brain regions with D > 0 had more CCK-GABA cells, whereas regions with D < 0 hadmore PV-GABA cells. Moving right along x-axis of the graph, a decreasing D reflects a relatively increasing PV-GABA cell content. Regions of the primary sensorycortex or primary motor cortex (red) had negative D, indicating relatively higher PV-GABA cell content. Interestingly, secondary regions (green) had less negative Dthan their primary counterparts, indicating relatively lower PV-GABA cell content. For comparison, other non-motor and non-sensory cortical subregionsare included (gray).

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to why CCK-GABA cells are more numerous in our modelis that GFP-labeling may not be entirely exclusive to CCK-GABA cells. Though GFP-labeling reflects the expression of theCCK transcript, some cells which express the CCK transcriptonly do so temporarily and do not actually produce the CCKpeptide (Tricoire et al., 2011). Therefore, a portion of GFP-labeled cells in this study may represent nonCCK-GABA cellsthat have a history of transient CCK expression. However,our specificity data suggests these nonCCK cells may onlyrepresent a modest portion of GFP-labeled cells (∼15%). Evenwhen excluding these cells from the CCK-GABA population,the adjusted percentage of CCK-GABA cells in many areas isstill much higher than that suggested by prior antibody labelingstudies. Accordingly, our data suggest that the population ofCCK-GABA cells has been at least slightly underestimated by theliterature.

The significance of the small interneuron populations (10–20% of all GABA cells) identified by the present study shouldnot be underestimated. Importantly, several studies reportthat even modest populations of perisomatic interneuronsmay serve important functions. The CCK-GABA cells ofthe basolateral amygdala, which likely constitute <10–15%of the total GABAergic cells in the region (unpublishedobservation), play a vital role in regulating fear and itsextinction (Bowers and Ressler, 2015). Other factors besidescell population size may be determinants of the functionalrelevance of an inhibitory network, such as the volume of post-synaptic connections exhibited by each cell. A single CCK- orPV-GABA cell, for example, may form synapses on thousandsof pyramidal cells. Given the ability of individual perisomaticinterneurons to influence a large proportion of the pyramidalcell network (Cobb et al., 1995), even small populations ofthese neurons may shape information processing and thereforebehavior.

The functional significance of the relatively large populationsof CCK-GABA cells within the medial prefrontal andventrolateral temporal cortices has not been determined.An attractive possibility is that these neuronal populationsmay help fine-tune working memory processes. CCK-GABAneurons commonly express CB1 receptors, which have beenconsistently implicated in working memory performancein humans and animals (Varvel and Lichtman, 2002; Hanet al., 2012; Ruiz-Contreras et al., 2014). Theoretical modelingsuggests that CCK-GABA neurons are ideally suited to regulateworking memory because they exhibit depolarization-inducedsuppression of inhibition (DSI), a form of plasticity mediatedby CB1 receptors (Wilson and Nicoll, 2001; Wilson et al., 2001).In CCK-GABA neurons, DSI limits the release of GABA fromthe presynaptic terminal (Wilson and Nicoll, 2001; Wilsonet al., 2001). Thus, DSI allows for CCK-GABA neurons todynamically adjust the strength of inhibitory transmissionto their post-synaptic targets, which include pyramidal cellsinvolved in working memory processing. Therefore, DSI mayallow pyramidal cells to maintain their activity – and presumablythe working memory representations that are encoded bytheir activity – for prolonged time periods (Carter and Wang,2007).

Interestingly, our comparative analysis suggested that theprimary and secondary areas of the cortex display a differentialbalance of PV- and CCK-GABA cells. In secondary corticalregions, the value of CCK-GABA cell percentage minus PV-GABA cell percentage was consistently higher than in primarycortical regions. Currently, the significance of this relativelygreater content of CCK-GABA cells to network activity isunclear. CCK-GABA cells may be particularly influential toinformation processing in these regions, as these cells mayintegrate signals over a prolonged timeframe (Bartos and Elgueta,2012) and maintain local activity patterns using a DSI-dependentmechanism (Carter andWang, 2007). This property is in keepingwith the nature of the secondary cortex, which integrates awealth of complex inputs from several sources. In contrast, CCK-GABA cells may be less effective for information processing inprimary cortex regions, which generally participate in the earlystages of sensory processing and must integrate and transmitsignals quickly. In this regard, fast-spiking PV-GABA cells maybe more advantageous (Jonas et al., 2004; Doischer et al.,2008). While the relative abundance of CCK-GABA cells inhuman cortex needs to be examined, their relative enrichmentwithin the secondary cortices of mice likely has importantimplications for human populations, as the relative size of thesecondary cortical areas is much greater in humans than inrodents.

The balance between CCK- and PV-GABA cells also hasimportant implications for network processing, as CCK-GABAcells may synapse with PV-GABA cells in areas such as thehippocampus (Hu et al., 2010; Lee and Soltesz, 2011). In thehippocampus, CCK-GABA cells release the peptide transmitterCCK on to adjacent PV-GABA cells, thereby exciting them (Leeand Soltesz, 2011). Thus, CCK-GABA neurons may act as a‘switch’ to initiate PV-GABA neuron activity. A loss of CCK-GABA neurons, such as that seen in neuropsychiatric disorders,may therefore disrupt the function of PV-GABA neurons andthe functions governed by these cells. Accordingly, a change inthe balance between CCK- and PV-GABA neurons may presentas a risk factor for behavioral impairment in disease. It remainsto be shown how the relationship between these neurons isaffected in pathology, as most studies examine a single neuronalpopulation.

The current study reveals a staggering array of GABAergiccell populations that currently have no identified function.The behavioral relevance of many large cell populations ofperisomatic interneurons, such as the PV-GABA cells of theretrosplenial cortex, has yet to be studied directly. In this regard,optogenetic approaches combined with the dual recombinase-based neuron strategy may be useful, as this method permitsthe selective activation of genetically distinct cells with a highdegree of spatial resolution. Optogenetic techniques have beenapplied to study of specific GABA neuron populations whichcontain somatostatin, vasoactive intestinal peptide and PV (Leeet al., 2013; Pi et al., 2013; Courtin et al., 2014), and couldbe of use in studying the interneuron populations shownhere. Besides optogenetics, selective activation of specific GABAneuron populations may also be possible using pharmacogenetictechniques such as ‘designer receptor exclusively activated by

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designer drug’ (DREADD) mouse model (Nguyen et al., 2014;Perova et al., 2015).

Our data support the validity of the dual recombinase-based approach as a tool for conducting quantitative analysisof neuronal subpopulations. Though only CCK- and PV-Frepetransgenic mouse lines were utilized in the present study, the dualrecombinase-based approach could also permit the visualizationof different cell types such as neuropeptide Y-, vasoactiveintestinal peptide- or reelin-expressing GABA neurons. Thisapproach would greatly facilitate the study of specific interneuronpopulations, many of which are still poorly characterized.Further, the dual recombinase-based genetic strategy can beapplied to study the role of specific GABA neurons in adiverse range of neural pathologies. Indeed, many disorders arecharacterized by profound changes in GABAergic cells or theirsignaling, including schizophrenia, maternal immune activation,stress, depression and autism (Peng et al., 2013; Perez and Lodge,2013; Aldrin-Kirk et al., 2014; Godavarthi et al., 2014; Kim et al.,2014a; Knoferle et al., 2014; Ma and McLaurin, 2014; Tong et al.,2014; Zamberletti et al., 2014). In these disorders, quantitativechanges in specific GABA neuron populations in discrete brainregions can be explored.

Author Contributions

PW and JK wrote the paper. JC managed the animal colony,prepared brain sections and conducted immunohistochemistry.

PW and NF collected all cell count data; PW performed allanalysis.

Funding

PW was funded by post-doctoral fellowships from the NaturalSciences and Engineering Council of Canada (NSERC) and theCanadian Institutes of Health Research (CIHR) through theSleep and Biological Rhythms Program. JC was supported by anOntario Graduate Scholarship. NF was supported by a NSERCUndergraduate Student Research Award. JK was supported by aNSERC Discovery grant (MOP 491009) and a CIHR grant (MOP496401).

Acknowledgments

The authors would like to thank Elena Soukhov for technicalassistance and Dr. John Yeomans for helpful comments.

Ethical Statement

All procedures were performed in accordance with the guidelinesof the National Institutes of Health (NIH) and the CanadianCouncil on Animal Care (CCAC) with approval from theUniversity of Toronto Animal Care Committee.

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

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